I. Membrane association of alpha-synuclein domains studied using VDAC nanopore reveals an unexpected binding pattern The exceptional diversity of interactions between peripheral membrane proteins and bilayer lipid membranes makes the membrane surface a rich scene for performing and regulating cellular functions. This same diversity presents significant experimental barriers to studies of interaction mechanisms among the components. One of the main challenges is that the energies of interaction between individual protein residues and lipid molecules are small; thus, statistical effects play a significant role. A variety of techniques to characterize binding of peripheral membrane proteins to liposome or planar lipid bilayer platforms have been developed, but it is increasingly clear that, except in the simplest of systems, no isolated technique yields a clear picture of the membrane binding process. In part, this is because these assays measure the average binding parameters of an ensemble of molecules, highlighting the need to characterize membrane-bound proteins at the single-molecule level. This year, we used the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane as a single-molecule probe for alpha-synuclein (a-syn), a protein of considerable clinical interest due to its well-established involvement in the pathology of Parkinson disease. It is well established that a-syn binding from solution to the surface of membranes composed of negatively charged and/or non-lamellar lipids can be characterized by equilibrium dissociation constants of tens of micromolar. However, we previously found that VDAC, reconstituted into planar bilayers of a plant-derived lipid, responds to a-syn at nanomolar solution concentrations. Now, using lipid mixtures that mimic the composition of mitochondrial outer membranes, we showed that functionally important binding does indeed take place in the nanomolar range. We demonstrated that the voltage-dependent rate at which a membrane-embedded VDAC nanopore captures a-syn is a strong function of membrane composition. Comparison of the nanopore results with those obtained by the bilayer overtone analysis of membrane binding demonstrated a pronounced correlation between the two datasets. The stronger the binding, the larger the on-rate, but with some notable exceptions. This leads to a tentative model of a-syn-membrane interactions, which assigns different lipid-dependent roles to the N- and C-terminal domains of a-syn accounting for both electrostatic and hydrophobic effects. As a result, the rate of a-syn capture by the nanopore is not simply proportional to the a-syn concentration on the membrane surface but found to be sensitive to the specific interactions of each domain with the membrane and nanopore. II. Real-time nanopore-based recognition of protein translocation success A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. This year we studied the translocation of a heterogeneously charged polypeptide using model-free detection of the effect of the polymer charge on the electrical environment inside the nanopore to monitor the translocation process of single polypeptides in real time. This was accomplished using selectivity tags regions of different but uniform charge density at the ends of a polypeptide that produce different selectivity of the nanopore to cations and anions, and hence ionic current levels, in a voltage-biased nanopore under a salt concentration gradient. We employed the natural, disordered diblock copolymer-like 140-amino-acid polypeptide alpha-synuclein, which comprises two such tags, a highly negatively charged C-terminal region (CT; 43 amino acids, total charge 15e) and a largely neutral N-terminal region (NT; 97 amino acids, total charge +3e). By using these features, we demonstrated a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The two selectivity tags at the ends of the polypeptide enabled us to discriminate between a-syn translocation and retraction. Our results demonstrated exquisite sensitivity of polypeptide translocation to the applied transmembrane potential and proved the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease. III. Mapping intra-channel diffusive dynamics of interacting molecules onto a two-site model: Crossover in flux concentration dependence Transport of various solutes through membrane channels is an extremely complex phenomenon. One of the reasons for this complexity is the interactions of solute molecules between themselves and with the channel. This year we addressed this problem by analyzing how these interactions affect the flux dependence on the solute concentration. The study focused on narrow membrane channels, where it was assumed that the molecules cannot bypass each other because of their hard-core repulsion. In addition, other short- and long-range solute-solute interactions were included into consideration. Such interactions make it impossible to develop an analytical theory for the flux in the framework of the continuum diffusion model of solute dynamics in the channel developed in our lab during recent years. To overcome this difficulty, we coarse-grained the diffusion model by mapping it onto a two-site one where the rate constants describing the solute dynamics were expressed in terms of the parameters of the initial diffusion model. This allowed us (i) to find an analytical solution for the flux as a function of the solute concentration and (ii) to characterize the solute-solute interactions by two dimensionless parameters. Such a characterization proved to be very informative as it resulted in a clear classification of the effects of the solute-solute interactions on the concentration dependence of the flux. Unexpectedly, it turned out that this dependence can be nonmonotonic, exhibiting a sharp maximum as a function of system parameters. In other words, we have found that the effect is quite nontrivial: The flux can reach a well-pronounced maximum at a certain optimal concentration, the value of which is defined by the interaction parameters. We hypothesize that this phenomenon may be used by nature as a sensory mechanism of a regulatory circuit, wherein an optimal solute concentration is reported upon by maximizing the transmembrane flux of the molecules.